Single turnover studies with oxy-cytochrome P-450 cam

Abstract

The catalytic step of bacterial cytochrome P-450 cam, i.e., the step of the reaction cycle in which the product 5-exo-hydroxycamphor is formed and released by the enzyme, has been studied by stopped-flow spectrophotometry. Our approach has been to observe a single-turnover reaction between reduced putidaredoxin and oxygenated camphor-bound cytochrome P-450 cam. Multiple turnovers are prevented by using the inhibitor metyrapone to trap the cytochrome after product release, which prevents binding of another camphor molecule. The time course of the reaction has been measured at several wavelengths and has been found to be biphasic. The relatively slow second phase of the reaction is the reduction of ferric, metyrapone-bound cytochrome P-450 cam. The first phase coincides with the formation of product stoichiometrically with cytochrome P-450 cam, as measured by gas chromatography. A detailed kinetic study of the first phase reveals a hyperbolic dependence of initial rate upon putidaredoxin concentration at a fixed, limiting concentration of cytochrome P-450 cam. The V max is 53 μ m per second per μ m cytochrome, and the K m for putidaredoxin is 33 μ m. The hyperbolic relationship between initial rate and putidaredoxin concentration supports a model in which the cytochrome rapidly binds putidaredoxin, then undergoes one or more slower intracomplex steps.